The cell wall of Gram-positive bacteria can be thought of as a microbial surface organelle with anchored proteins that require specific targeting mechanisms for their assembly, function and release. For example, sortase A recognizes the LPXTG motif within sorting signals of secreted proteins, which are cut and covalently linked to peptidoglycan. In Staphylococcus aureus, an important human pathogen, two types of surface protein precursors are distinguished. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with YSIRK-G/S motif signal peptides traffic to the cross wall. The cross wall separates newly divided daughter cells and is split along its central axis to complete the cell cycle. In this proposal, the trafficking and function of two surface proteins are examined with the goal of revealing new principles of bacterial pathogenesis. SpA, an immunoglobulin-binding surface protein with YSIRK-G/S motif, traffics to the cross wall. Following cross wall splitting and cell separation, SpA is distributed on the bacterial surface and blocks antibody-induced opsonophagocytic killing of staphylococci. Through the catalytic action of specific murein hydrolases, SpA is released from the surface organelle and is proposed to activate B cell receptors and modulate antibody development in infected hosts. AdsA, a surface protein with a canonical signal peptide, is deposited at the cell pole and is also released from the staphylococcal surface. We hypothesize that AdsA converts adenine nucleotides (AMP, ADP & ATP) to adenosine (Ado), which suppresses host immune functions via adenosine receptor signaling. Further, AdsA, together with nuclease, generates deoxyadenosine (dAdo) from neutrophil NETs to induce macrophage apoptosis. Staphylococci may secrete additional products that interfere with Ado and dAdo deamination during infection. Here we propose to study the mechanisms whereby SpA and AdsA traffic to specific sites in the cell wall envelope and are released from the bacterial surface. We also propose to study the molecular biology of staphylococcal Ado/dAdo and B cell receptor signaling in infected hosts and examine small molecule inhibitors of adenosine receptors for their therapeutic impact on staphylococcal disease in mice.